Characterization of primary direct-acting antiviral (DAA) drugs resistance mutations in NS5A/NS5B regions of hepatitis C virus with genotype 1a and 1b from patients with chronic hepatitis

ABSTRACT The Hepatitis C virus (HCV) infection is a public health problem. The high level of HCV replication and its lack of post-transcriptional correction mechanisms results in the emergence of viral variants and the difficulty in determining polymorphisms and variants that contain the substitutions associated with resistance towards new antivirals. The main focus of this study was to map the NS5A and NS5B polymorphisms and resistance mutations to new antiviral drugs in HCV strains genotype 1 from patients with chronic hepatitis C infection. Serum samples were collected from patients who underwent routine viral load tests at the Instituto Adolfo Lutz, Sao Paulo city, Brazil. A total of 698 and 853 samples were used for the characterization of NS5A and NS5B regions, respectively, which comprise the HCV genotypes 1a and 1b. The prevalence of resistance mutations found in the NS5A region was 6.4%, with Y93H, L31M, Q30R, and Y93N as the main resistance-associated substitutions (RAS). No NS5B-associated RAS was observed for any of the analyzed drugs. These findings support that the RAS test should be offered to individuals with poor response to double combination regimens prior to treatment initiation, thereby assisting strain vigilance and selection of effective treatment or retreatment options using DAA regimens.

Phytochemical analysis of Berberis lyceum methanolic extract and its antiviral activity through the restoration of MAPK signaling pathway modulated by HCV NS5A

Objective: To evaluate the antiviral activity and phytochemicals of selected plant extracts and their effect on the mitogen-activated protein kinase (MAPK) signaling pathway modulated by hepatitis C virus (HCV) nonstructural protein 5A (NS5A). Methods: A total of ten plant extracts were initially screened for their toxicities against HepG2 cells. The non-toxic plants were tested for their inhibitory effect on the expression of HCV NS5A at both mRNA and protein levels using real-time PCR and Western blotting assays, respectively. The differential expression of the genes associated with MAPK pathway in the presence of NS5A gene and plant extract was measured through real-time PCR. Subsequently, the identification of secondary metabolites was carried out by phytochemical and HPLC analysis. Results: The phytochemical profiling of Berberis lyceum revealed the presence of alkaloids, phenols, saponins, tannins, flavonoids, carbohydrates, terpenoids, steroids, and glycosides. Similarly, quercetin, myricetin, gallic acid, caffeic acid, and ferulic acid were identified through HPLC analysis. The methanolic extract of Berberis lyceum strongly inhibited HCV RNA replication with an IC50 of 11.44 μg/mL. RT-PCR and Western blotting assays showed that the extract reduced the expression of HCV NS5A in a dose-dependent manner. Berberis lyceum extract also attenuated NS5A-induced dysregulation of the MAPK signaling pathway. Conclusions: Our findings suggest that Berberis lyceum extract strongly inhibits HCV propagation by reducing HCV NS5A-induced perturbation of MAPK signaling.

Phytochemical analysis of Berberis lyceum methanolic extract and its antiviral activity through the restoration of MAPK signaling pathway modulated by HCV NS5A

Objective: To evaluate the antiviral activity and phytochemicals of selected plant extracts and their effect on the mitogen-activated protein kinase (MAPK) signaling pathway modulated by hepatitis C virus (HCV) nonstructural protein 5A (NS5A). Methods: A total of ten plant extracts were initially screened for their toxicities against HepG2 cells. The non-toxic plants were tested for their inhibitory effect on the expression of HCV NS5A at both mRNA and protein levels using real-time PCR and Western blotting assays, respectively. The differential expression of the genes associated with MAPK pathway in the presence of NS5A gene and plant extract was measured through real-time PCR. Subsequently, the identification of secondary metabolites was carried out by phytochemical and HPLC analysis. Results: The phytochemical profiling of Berberis lyceum revealed the presence of alkaloids, phenols, saponins, tannins, flavonoids, carbohydrates, terpenoids, steroids, and glycosides. Similarly, quercetin, myricetin, gallic acid, caffeic acid, and ferulic acid were identified through HPLC analysis. The methanolic extract of Berberis lyceum strongly inhibited HCV RNA replication with an IC50 of 11.44 μg/mL. RT-PCR and Western blotting assays showed that the extract reduced the expression of HCV NS5A in a dosedependent manner. Berberis lyceum extract also attenuated NS5Ainduced dysregulation of the MAPK signaling pathway. Conclusions: Our findings suggest that Berberis lyceum extract strongly inhibits HCV propagation by reducing HCV NS5Ainduced perturbation of MAPK signaling.

Mode of Oral Ethanol Feeding Affects Liver Oxidative Stress Levels and Methylation Status: Study on NS5A-Transgenic Mice.

Alcohol consumption accelerates the progression and worsens the outcomes of hepatitis C viral (HCV) infection in heavy and moderate drinkers. Our aim was to investigate the effects of two modes of oral ethanol feeding on induction of oxidative stress, impaired methylation status and downstream changes in proteasome activity in livers of NS5A-transgenic (Tg) mice. Methods: Ethanol was administered either in water (chow fed mice given 20% ethanol in water; designated chow-EtOH) or fed in Lieber De Carli liquid diet (LCD-EtOH). Appropriate controls were used. The mechanisms of alcohol and NS5A-induced changes in oxidative stress, liver methylation status and 20S proteasome activity were determined after 5 weeks of the feeding regimen. Results: Ethanol administration using both feeding regimens induced oxidative stress and suppressed cytosolic proteasome activity. However, only LCD-EtOH diet induced fatty changes in the liver which correlated with higher levels of oxidative stress, impaired methylation potential and reduced cytosolic and nuclear proteasome activity. However, LCD diet by itself triggered lipid peroxidation. Conclusion: We conclude that both modes of oral ethanol feeding (chow and LCDbased) induce oxidative stress in NS5A-Tg mice that suppresses proteasome activity. Nonetheless, impaired methylation potential, higher level of oxidative stress and suppression of nuclear proteasome was observed only in LCD-EtOH mice. However, the effects of LCD-control liquid diet in inducing lipid peroxidation in NS5A-Tg mice, in certain cases, tended to mask the effects of ethanol.

Screening of cellular proteins that interact with the classical swine fever virus non-structural protein 5A by yeast two-hybrid analysis.

Classical swine fever virus (CSFV), the pathogen of classical swine fever (CSF), causes severe hemorrhagic fever and vascular necrosis in domestic pigs and wild boar. A large number of evidence has proven that non-structural 5A (NS5A) is not only a very important part of viral replication complex, but also can regulate host cell’s function; however, the underlying mechanisms remain poorly understood. In the current study, aiming to find more clues in understanding the molecular mechanisms of CSFV NS5A’s function, the yeast two-hybrid (Y2H) system was adopted to screen for CSFV NS5A interactive proteins in the cDNA library of the swine umbilical vein endothelial cell (SUVEC). Alignment with the NCBI database revealed 16 interactive proteins: DDX5, PSMC3, NAV1, PHF5A, GNB2L1, CSDE1, HSPA8, BRMS1, PPP2R3C, AIP, TMED10, POLR1C, TMEM70, METAP2, CHORDC1 and COPS6. These proteins are mostly related to gene transcription, protein folding, protein degradation and metabolism. The interactions detected by the Y2H system should be considered as preliminary results. Since identifying novel pathways and host targets, which play essential roles during infection, may provide potential targets for therapeutic development. The finding of proteins obtained from the SUVEC cDNA library that interact with the CSFV NS5A protein provide valuable information for better understanding the interactions between this viral protein and the host target proteins.

Recent advance in antiviral drugs for hepatitis C / 中南大学学报(医学版)

Hepatitis C virus (HCV) infection is the leading cause of chronic liver diseases worldwide.There is no vaccine to prevent HCV infection.Current standard of care (SOC) for hepatitis C is pegylated interferon-α (pegIFN-α) in combination with ribavirin (RBV).However,the efficacy of pegIFN-α and RBV combination therapy is less than 50％ for genotype 1 HCV,which is the dominant virus in human.Additionally,IFN and RBV are highly toxic,causing severe side effects.Therefore,it is urgent to develop safer and more efficacious anti-HCV drugs.Over the last decade,a number of HCV-specific inhibitors have been discovered with many of them reached to late stages of clinical trials.Recently,2 HCV NS3 protease inhibitors,telaprevir and boceprevir,have been approved by the Unite States Food and Drug Administration (FDA).This opens up a new era for anti-HCV therapy.Several new classes of antiviral drugs targeting HCV NS3 protease,NS5A and NSSB RNA-dependence RNA polymerase (RdRp) are currently at various stages of preclinical and clinical studies.Upon approval of more NS3 protease,NS5A and NS5B polymerase inhibitors,future clinical studies will lead to optimal combination therapies which will have desirable parameters such as IFN-free,higher efficacy,safe,one daily dose and short duration.

Sequencing of E2 and NS5A regions of HCV genotype 3a in Brazilian patients with chronic hepatitis

Hepatitis C virus (HCV) is a major cause of liver disease throughout the world. The NS5A and E2 proteins of HCV genotype 1 were reported to inhibit the double-stranded (ds) RNA-dependent protein kinase (PKR), which is involved in the cellular antiviral response induced by interferon (IFN). The response to IFN therapy is quite different between genotypes, with response rates among patients infected with types 2 and 3 that are two-three-fold higher than in patients infected with type 1. Interestingly, a significant percentage of HCV genotype 3-infected patients do not respond to treatment at all. The aim of this paper was to analyse the sequences of fragments of the E2 and NS5A regions from 33 outpatients infected with genotype 3a, including patients that have responded (SVR) or not responded (NR) to treatment. HCV RNA was extracted and amplified with specific primers for the NS5A and E2 regions and the PCR products were then sequenced. The sequences obtained covered amino acids (aa) 636-708 in E2 and in NS5A [including the IFN sensitivity determining region (ISDR), PKR-binding domain and extended V3 region)]. In the E2 and NS5A regions, we did observe aa changes among patients, but these changes were not statistically significant between the SVR and NR groups. In conclusion, our results suggest that the ISDR domain is not predictive of treatment success in patients infected with HCV genotype 3a.

Progress in Research of the Role of Hepatitis C Virus Protein in Cell Signal Transduction Pathway / 中国实验动物学报

Abnormal cell signal transduction is associated with the occurrence and development of human diseases. Some virus pathogenicity and infection mechanism are due to virus antigen protein acting on the host cell signal transduction pathway, leading to host cell signal transduction disorder. Hepatitis C virus (HCV) is a major pathogen of chronic hepatitis C, which causes cirrhosis and hepatocellular carcinoma. But the pathogenesis of HCV and persistent infection mechanism remain far from clear. HCV pathogenesis may be related to the HCV protein expression interfering host cell signal transduction pathways. The studies of hepatitis C virus proteins acting on host cell signal transduction pathways, not only help to clarify the impact of its pathogenic mechanisms, but also benefit to new drug design and development for new treatment methods. This article summarizes the recent progress in research on the effect of hepatitis C virus protein in cell signal transduction pathways in the past few years.

Genetic variability in the 5' UTR and NS5A regions of hepatitis C virus RNA isolated from non-responding and responding patients with chronic HCV genotype 1 infection

Sequence variation among different hepatitis C virus (HCV) isolates has adaptive significance and reflects the modes and intensities of selection mechanisms operating on the virus. In this work, we sought to investigate using classical population genetics parameters, the genetic variability of HCV genotype 1 using the 5' UTR and NS5A regions from treatment non-responding and responding groups of patients. Both regions showed low genetic varia-bility and the 5' UTR showed neutral deviation. No differences were observed in the nonsynonymous/synonymous nucleotide substitution ratio among groups for NS5A. The analysis of molecular variance test of the 5' UTR region showed an 11.94 percent variation among groups. Phylogenetic analysis showed no correlation between sequence variations and therapeutic responses.

Construction of eukaryotic expression vector of hepatitis C virus NS5A gene / 吉林大学学报(医学版)

Objective To construct an eukaryotic expression vector of the hepatitis C virus(HCV)NS5A gene.and obtain a stable transfected Huh-7 cell line which provides a basis for further investigation of the hepatitis virus C NS5A protein.Methods The HCV NS5A gene from the pcDNA3.1(+)/HCV NS345 plasmid with HCV NS5A gene was amplified by PCR,and cloned to pGEM-T vector,and transformed into E.coli JM109.The positive colonies were first confirmed by restriction enzyme digestion and sequencing and then were inserted to eukaryotic expression vector pCI-neo,and verified by enzyme digestion and sequencing.Results After TA colon of the HCV NS5A was amplified by PCR,the positive colonies were finally verified by sequencing,which were totally in line with the designed coding sequence of HCV NS5A gene.Conclusion Eukaryotic expression vector of HCV NS5A gene has been successfully constructed.

Prokaryotic expression and bioinformatics analysis of NS5A transactivating protein 7 of hepatitis C virus / 解放军医学杂志

Objective To construct prokaryotic expression vector of NS5A transactivating protein 7 of hepatitis C virus (NS5ATP7), induce the expression of NS5ATP7 in E. coli, and to predict its structure and function by bioinformatics analysis. Methods NS5ATP7 gene was amplified by reverse transcription PCR (RT-PCR) using HepG2 cells mRNA as template, and was ligated into pGEM-T cloning vector. After verifying the sequence of the inserted fragment by restriction enzyme digestion identification and sequencing, NS5ATP7 was cloned into inducible prokaryotic expression vector pET-32a(+) and transfected into competent E. coli BL21. The protein expression was induced with IPTG and the product was analyzed by SDS-PAGE and Western blotting hybridization. The structure and function of NS5ATP7 were predicted using bioinformatics techniques. Results NS5ATP7 gene with about 891bp was amplified by RT-PCR, which was coincident with that we expected, and it was then inserted into expression vector pET-32a(+). Under the induction of IPTG, the recombinant NS5ATP7 was expressed successfully. SDS-PAGE and Western blotting assay showed that this recombinant protein possessed good immunogenicity. Bioinformatics method such as ExPASy, TMHMM and SignalP analysis indicated that NS5ATP7 was full of helices, had neither transmembranous structure nor signal peptide. Conclusions The recombinant NS5ATP7 gene could be successfully expressed in prokaryotic expression system of E. coli, which might lay the foundation of studying the immunogenicity and biological charactersitics of the NS5ATP7. Bioinformatics analysis may provide a new method to analyze the structure and function of a new protein.

CLONING OF THE HEPATITIS C VIRUS NONSTRUCTURAL PROTEIN 5A BINDING PROTEIN 37 CODING GENE / 解放军医学杂志

The nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) has been shown to interact with a variety of cellular proteins and implicated in the regulation of cell growth, interferon resistance, and other cellular signaling pathways. Using the yeast-two hybrid method, we have isolated a clone that encodes a novel NS5A--associated binding protein: NS5ABP37. Reverse transcription polymerase chain reaction (RT-PCR) method was employed to amplify the full fragment,and the plasmid pGADT7-NS5ABP37 with the Saccharomyces cerevisiae vector pGADT7 was constructed. To prove the interaction, yeast cell Y187 transformed with pGADT7-NS5ABP37 was mated with yeast cell AH109 containing pGBKT7-NS5A to verify the interaction between the novel protein coded by the new gene NS5ABP37 and NS5A.

Cloning and identification of human gene 3 transactivated by nonstructural protein 5A of hepatitis C virus / 解放军医学杂志

Objective To screen and clone the target genes transactivated by nonstructural protein 5A (NS5A) of hepatitis C virus (HCV). Methods The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-NS5A and pcDNA3.1(-) empty vector, respectively. Suppression subtractive hybridization (SSH) method was employed to analyze the differentially expressed DNA sequence between the two groups. The obtained sequences were searched for homologous DNA sequence from GenBank. The new gene with no homology with known genes in this database was confirmed, and electric polymerase chain reaction was conducted for cloning the full-length DNA of the new gene and in conjunction with Kozak rule and the terminus of polyadenyl signal sequence. The reverse transcription PCR (RT-PCR) was used to amplify the new gene from mRNA of HepG2 cell as the template. The coding sequence of the new gene was deduced according to the nucleotide sequence. Results A new gene with unknown function was named as NS5ATP3. The nucleotide sequence of the NS5ATP3 gene and its corresponding amino acid have been determined, which contained 1 572nt and 524aa. The sequence of the NS5ATP3 gene was deposited into GenBank, with the accession number AF529364. Conclusions NS5ATP3 gene transactivated by HCV NS5A protein was cloned and identified successfully by combining molecular biological technology and bioinformatics technique. These results will pave the way for the study of the molecular mechanism of the transactivating effects of HCV NA5A protein and the development of new therapy for chronic hepatitis C.

SUPPRESSION SUBTRACTIVE HYBRIDIZATION FOR CLONING OF GENES TRANSACTIVATED BY NS5A PROTEIN OF HEPATITIS C VIRUS / 解放军医学杂志

To construct a subtractive cDNA library of genes transactivated by NS5A protein of hepatitis C virus with suppression subtractive hybridization technique, the mRNA was isolated from HepG2 cells transfected with pcDNA3 1(-) NS5A and pcDNA3 1(-) empty vector, respectively, then the cDNA was synthesized. After restriction enzyme RsaI digestion, small sized cDNAs were obtained. Then the tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After the tester cDNA was hybridized with the driver cDNA twice and underwent two times of nested PCR, it was then subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain JM109. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. The amplified library contained 121 positive clones. Colony PCR showed that 115 clones contained 200- 1 000 bp inserts. Sequence analysis was performed in 90 clones, and the full length sequences were obtained with bioinformatics method. Altogether 46 kinds of coding sequences were acquired, which consisted of 31 kinds of known and 15 kinds of unknown ones. The obtained sequences might be target genes transactivated by NS5A protein of HCV, among which some genes coding proteins involved in cell cycle regulation, cell apoptosis, signal transduction pathway and tumour development. The results indicated that the subtractive library of genes transactivated by NS5A protein of HCV was constructed successfully, which brought some new clues for studying the biological functions and pathogenesis of the viral proteins

SCREENING AND CLONING OF THE GENES OF PROTEIN INTERACTING WITH THE NONSTRUCTURAL PROTEIN 5A OF HEPATITIS C VIRUS / 解放军医学杂志

Protein protein binding is the basis of virus and host cell interactions. With the application of technology of studying of protein interactions, more knowledge of replication and pathogenesis of hepatitis C virus (HCV) could be acquired. Non structure protein NS5A is one of the important regulatory factors in virus replication , transcription and signal transduction, but there are controversy in effect on HCV pathogenesis and resistance to interferon ?(IFN?). In order to describe the relationship between NS5A and host proteins, we use yeast two hybrid system 3 to screen the gene encoding proteins that could interact with NS5A from human hepatocyte library. Thirty five clones were obtained including apo A1, apo A2, apo B100, haplotype mitochondrion complete genome, phosphatidic acid phosphatase type 2B, albumin similar to tumor endothelial marker 5 precursor, matrix metalloproteinase 14, and three of the Homo sapiens hypothetical proteins. The study paved a way for further studies on the pathogenesis of HCV NS5A

Cloning and expression of ns5a gene of hepatitis C virus 1b strain DY in Escherichia coli / 西安交通大学学报(医学版)

Objective To clone and express the ns5a gene of hepatitis C virus(HCV) 1b strain DY.Methods By using the prokaryotic cell gene engineering,HCV ns5a gene was amplified with nested PCR from the plasmid HCV17 of HCV 1b strain DY full-length gene and inserted into the cloning pMD18-T vector.The cloned HCV ns5a gene was separated and subcloned into expression vector pET-28a and induced by IPTG in E.coli.BL21.The expressed product was identified by SDS-PAGE and Western-blot methods.Results Recombinant expression plasmid pet-28a-ns5a was constructed and expressed successfully.Conclusion HCV ns5a gene was cloned and expressed.This might be helpful for further studies on the nature and biological properties of the ns5a gene.